Suppression of intestinal carcinogenesis in Apc mutant mice ... - J-Stage

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Jun 17, 2015 - 2Graduate School of Life Sciences, Toyo University, 1 1 1 Izumino, Itakura ..... Grants-in-Aid for Project Future, Relay for Life (Japan Cancer.
Original Article Journal JCBN the 1880-5086 0912-0009 Kyoto, Original 10.3164/jcbn.15-28 jcbn15-28 Society Japan ofArticle Clinical for FreeBiochemistry Radical Research and Nutrition Japan Suppression of intestinal carcinogenesis in Apcmutant mice by limonin Satomi Shimizu,1,2 Shingo Miyamoto,1 Gen Fujii,1 Ruri Nakanishi,1 Wakana Onuma,1 Yoshihiko Ozaki,3 Kyoko Fujimoto,4 Tomohiro Yano2 and Michihiro Mutoh1,* 1 Epidemiology and Prevention Division, Research Center for Cancer Prevention and Screening, National Cancer Center, 511 Tsukiji, Chuoku, Tokyo 1040045, Japan 2 Graduate School of Life Sciences, Toyo University, 111 Izumino, Itakuramachi, Ogagun, Gunma 3740193, Japan 3 Faculty of BiologyOriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa City, Wakayama 6496493, Japan 4 Division of Molecular Biology, Nagasaki International University, 28257 Huis Ten Bosch, Sasebo, Nagasaki 859 3298, Japan

(Received 17 February, 2015; Accepted 4 March, 2015; Published online 17 June, 2015) ??

Limonoids in open citrus fruits known to possess multiple biological Creative stricted vided Copyright This 2015 the isuse, original an Commons distribution, © 2015 work access JCBN Attribution isare article and properly reproduction distributed License, cited. under which in any the permits medium, terms of unreprothe functions, such as antiproliferative functions in human cancer cell lines. Therefore, we aimed to investigate the suppressive effect of limonin on intestinal polyp development in Apcmutant Min mice. Fiveweekold female Min mice were fed a basal diet or a diet containing 250 or 500 ppm limonin for 8 weeks. The total number of polyps in mice treated with 500 ppm limonin decreased to 74% of the untreated control value. Neoplastic cell proliferation in the polyp parts was assessed by counting PCNA positive cells, and a tendency of reduction was obtained by limonin treatment. More over, expression levels of cMyc and MCP1 mRNA in the polyp part were reduced by administration of limonin. We finally con firmed the effects of limonin on βcatenin signaling, and found limonin significantly inhibited Tcell factor/lymphocyte enhancer factordependent transcriptional activity in a dosedependent manner in the Caco2 human colon cancer cell line. Our results suggest that limonin might be a candidate chemopreventive agent against intestinal carcinogenesis. Key Words:

limonin, Min mice, colon cancer, cMyc, Tcf/Lef

C

Colorectal cancer is the third most common cancer in the Introduction world. Nearly 1.4 million new cases occurred in 2012, and this is expected to increase to 2.4 million annually by 2035 worldwide.(1) Despite extensive efforts to develop many anti-cancer drugs, mortality of colorectal cancer is still high. Therefore, a new strategy for controlling development of cancer is in great demand.(2) One approach is the use of functional agents in foods or plants as supplements or adjuvant agents in chemotherapy.(3,4) The other approach is chemoprevention for the inhibition of colorectal cancer development.(3,4) Since epidemiological studies suggest that a high intake of fruit is associated with a reduced risk of chronic disease, including cancer,(5) using functional agents in citrus fruits seems to be an attractive approach for both methods. Citrus fruits are good resources for a variety of constituents important to human nutrition, such as vitamin C, folic acid, potassium, flavonoids, pectin, and dietary fiber. Citrus also contains highly oxygenated triterpenoids (limonoids), which contain a furan ring attached to the D-ring at C-17 as well as an epoxide group at C-14 and C-15, and oxygen containing functional groups at C-3, C-4, C-7, C-16 and C-17. Limonoids were firstly identified as components contributing to bitterness in citrus juices.(6) Recently, it is has been revealed that limonoids possess multiple biological functions in vitro and in vivo.(7) Notably, limonin, the most abundant limonoid in citrus fruits, showed anti-proliferative efficacy in several human cancer cell lines(8–10) and a suppressive effect on the development of aberrant crypt foci (ACF) in AOM-

doi: 10.3164/jcbn.1528 ©2015 JCBN

treated rats.(11) Although several reports have shown that limonin exhibits anticancer effects in vitro and suppressive effects on the formation of preneoplastic lesions in vivo, the effects of limonin on intestinal carcinogenesis have not been addressed. In this study, we investigated the impact of limonin on intestinal polyp formation in Apc-mutant Min mice, an animal model of human familial adenomatous polyposis that develops numerous polyps in the intestinal tract. We also evaluated the effects of limonin on the transcriptional activity of the Wnt signaling pathway, which plays an important role in the proliferation of cancer cells, in a human colon cancer cell line. Materials and Methods Cell cultures and chemicals. Caco-2 cells, a human colon adenocarcinoma cell line, were purchased from Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan). The cells were maintained in DMEM medium supplemented with 10% heatinactivated fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT), MEM Non-Essential Amino Acids Solution (Nacalai Tesque, Inc., Japan) and antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin) at 37°C in 5% CO2. Limonin for animal study was isolated from grapefruit seed, and kindly provided by Dr. Shin Hasegawa (USDA, ARS, WRRC, retired). Limonin for cell culture study was purchased from Wako (Osaka, Japan). Animals. Female C57BL/6-ApcMin/+ mice (Min mice) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed per plastic cage with sterilized softwood chips as bedding in a barrier-sustained animal room at 24 ± 2°C and 55% humidity on a 12 h light/dark cycle. Limonin was well mixed at a concentration of 250 or 500 ppm in AIN-76A powdered basal diet (CLEA Japan, Tokyo, Japan). Protocol for animal experiments. Ten female Min mice at 5 weeks of age were given 250 or 500 ppm limonin for 8 weeks. The animals in each cage were all in the same treatment group. Food and water were available ad libitum. The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured weekly. The intestinal tract was removed and separated into the small intestine, cecum and colon. The small intestine was divided into the proximal segment (4 cm in length) from the stomach and then the proximal (middle) and distal halves of the remaining part. Polyps in the proximal segments were counted immediately, and all polyps in the proximal *To whom correspondence should be addressed. Email: [email protected]

J. Clin. Biochem. Nutr. | July 2015 | vol. 57 | no. 1 | 39–43

Table 1. Number of intestinal polyps/mouse in Min mice Limonin (ppm)

No. of mice

Small intestine Proximal

Middle

Distal

Colon

Total

0

10

4.9 ± 2.1

9.7 ± 3.2

18.0 ± 7.4

0.0 ± 0.0

35.2 ± 9.4

250

10

3.8 ± 2.0

8.9 ± 2.6

16.3 ± 6.3

0.2 ± 0.4

29.2 ± 5.4

500

10

3.6 ± 2.6

8.7 ± 3.0

13.2 ± 3.3*

0.1 ± 0.3

25.6 ± 5.2*

Data are mean ± SD. *Significantly different from the control untreated group at p